POS0344 O-GlcNAcylation ON NUP153 REGULATES THE EARLY STAGES OF OSTEOCLASTOGENESIS THROUGH MYC NUCLEAR TRANSLOCATION
Authors: Y. N. LI, C. W. Chen, T. Trinh-Minh, Z. Honglin, P. Hubel, J. Pfannstiel, G. Schett, J. H. W. Distler
Journal: Annals of the Rheumatic Diseases (2022)

Background Bone homeostasis is maintained by the balance between bone formation and resorption. In inflammatory arthritis, such as rheumatoid arthritis (RA), the pro-inflammatory environment promotes osteoclast differentiation and skews this balance towards bone resorption, leading to destructive bone erosion and bone loss. O-GlcNAcylation is one of the most common post-translational modifications, which attaches a single N-acetylglucosamine (GlcNAc) molecular to the serine or threonine of the target protein. O-GlcNAcylation is controlled by the activities of a single pair of enzymes: OGT, which facilitates the transfer of GlcNAc onto proteins; OGA, which removes GlcNAc from proteins. The activity of O-GlcNAcylation has been reported to be involved in several cellular events, such as transcription, translation, intracellular trafficking, and differentiation. We previously showed that the dynamics of O-GlcNAcylation are essential for osteoclast differentiation. TNF-α, a key pro-inflammatory factor in RA, intensified the O-GlcNAcylation dynamics. Inhibition of OGT arrests osteoclast precursors at early stages, whereas OGA inhibition blocks osteoclast maturation. However, the molecular mechanism of these regulations remains unclear. Objectives We aimed to identify the O-GlcNAcylation targets in osteoclast precursors in a pro-inflammatory milieu and to decipher the molecular mechanism of O-GlcNAcylation mediated regulation of osteoclastogenesis. Methods We first identify the O-GlcNAc-dependent molecular pathways in osteoclast precursors with pharmacological OGT and OGA inhibition by RNA sequencing. Then, we identified the O-GlcNAcylated proteins by mass spectrometry-based proteomics analysis and confirmed by immunoprecipitation. We found the potential molecular mechanism by combining the data from transcriptomics and proteomics. The proposed mechanism was further validated through siRNA-mediated knockdown and high-content screening analysis. Results Our transcriptomics data showed that OGT inhibition arrested osteoclast differentiation at early stages through interfering the cytokine signaling and metabolic adaption. The upstream analysis proposed MYC as the most potent regulator for the transcriptomic profile under OGT inhibition. Recent studies proposed MYC as a master regulator for metabolic reprograming during osteoclast differentiation. However, O-GlcNAcylation of MYC was not detected by mass spectrometry, suggesting indirect effects of O-GlcNAcylation on MYC signaling in osteoclast precursors. We detected upregulated levels of O-GlcNAc on NUP153, MTDH, RBM27, IFI207 upon RANKL+TNFα stimulation. An integrated analysis of transcriptomic and proteomic data by Ingenuity Pathway Analysis indicated that NUP153 might regulate the most DEGs among all the identified targets and indicated potential of NUP153 to regulate nuclear shuttling of MYC. Subcellular fractionation and confocal microscopy showed enhanced MYC nuclear translocation upon RANKL+TNFα stimulation, which could be blocked by NUP153 knockdown or OGT inhibition. Functionally, knockdown of NUP153 arrested cells at similar stages to OGT inhibition and reduced bone resorption ability. Together, these results suggest a model, in which O-GlcNAcylation regulates the shuttling activity of the nuclear pore component NUP153 to control the access of MYC to the nucleus during osteoclast differentiation. Conclusion Our results indicated that OGT inhibition arrests osteoclastogenesis at early stages through hampering MYC-dependent metabolic adaption. NUP153 was proposed as the most potent O-GlcNAcylation target by multi-omics data integration. NUP153-mediated MYC nuclear trafficking is required for osteoclast differentiation. These findings reveal the molecular mechanism of O-GlcNAcylation-dependent osteoclastogenesis and provide therapeutic insights on targeting O-GlcNAcylation in pathologic bone resorption. Disclosure of Interests Yi-Nan Li: None declared, Chih-Wei Chen: None declared, Thuong Trinh-Minh: None declared, ZHU Honglin: None declared, Philipp Hubel: None declared, Jens Pfannstiel: None declared, Georg Schett: None declared, Jörg H.W. Distler Shareholder of: 4D Science, Speakers bureau: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Employee of: FibroCure